This research involves a combined cytogenetic, immunological and molecular approach to the role of mammalian ribosomal RNA gene amplification in viral and chemical carcinogenesis and tumor progression. Q-, G-, and R-banding will be used to identify probable amplified regions in developing tumors or tumor cell lines, AgNOR staining to detect actively transcribed rRNA genes, antibodies to 5-methylcytidine and an indirect immunoperoxidase technique to detect chromosome regions highly enriched in 5-methylcytosine, and in situ hybridization to radiolabeled 5S, 18S and 28S RNA probes to show the location and multiplicity of these coding sequences in chromosome segments and in restriction endonuclease fragments. Hpa II and Msp I digestion of amplified hypermethylated DNA will be used, in conjunction with digestion by other enzymes, to map the sites of methylation that are potentially important in gene regulation.